Large volume ex vivo electroporation method

ABSTRACT

An object of the invention is to provide an electroporation method for treating vesicles with exogenous material for insertion of the exogenous material into the vesicles which includes the steps of: a. retaining a suspension of the vesicles and the exogenous material in a treatment volume in a chamber which includes electrodes, wherein the chamber has a geometric factor (cm −1 ) defined by the quotient of the electrode gap squared (cm 2 ) divided by the chamber volume (cm 3 ), wherein the geometric factor is less than or equal to 0.1 cm −1 , wherein the suspension of the vesicles and the exogenous material is in a medium which is adjusted such that the medium has conductivity in a range spanning 50 microSiemens/cm to 500 microSiemens/cm, wherein the suspension is enclosed in the chamber during treatment, and b. treating the suspension enclosed in the chamber with one or more pulsed electric fields. With the method, the treatment volume of the suspension is scalable, and the time of treatment of the vesicles in the chamber is substantially uniform.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to the following applications that areincorporated by reference herein: U.S. Provisional Patent Application,of Walters and King, Application No. 60/454,360, Filing Date 14 Mar.2003, for LARGE VOLUME EX VIVO ELECTROPORATION METHOD; PCT Applicationof Walters and King Application Number PCT/US04/05237 filed on Mar. 15,2004 for LARGE VOLUME EX VIVO ELECTROPORATION METHOD; and U.S.Non-provisional Application of Walters and King, application Ser. No.10/537,254 filed on Jun. 1, 2005 for LARGE VOLUME EX VIVOELECTROPORATION METHOD.

BACKGROUND OF THE INVENTION Technical Field

The present invention relates generally to ex vivo electroporationmethods, and, more particularly, to electroporation methods especiallyadapted for clinical and industrial applications.

Background Art

Delivering large molecules into living cells for therapeutic purposes,using ex vivo or in vitro electroporation, has been described in theliterature for many years. The purpose of electroporation is to enhancethe movement of molecules into and out of living cells or non-livingvesicles. The practical uses are many and vary according to thecomplexity of material delivered, the site of delivery and the purposefor delivery.

Complexity ranges from small drug molecules that are otherwise difficultto get into cells to complex mixtures of polynucleotides.

The site of delivery is broadly divided into in vivo and ex-vivodelivery. The choice of an in-vivo site is based upon the location ofthe tissue to be treated and whether or not local or systemic treatmentis desired.

Clinical and industrial applications of this process are possible.Often, in clinical and industrial applications, it is desirable toinsert large molecules into large numbers of cells and to insure thatall cells have been processed equally. To do that, it is desirable toprocess all cells simultaneously to guarantee that all cells aresubjected to the same process conditions.

Therapeutic purposes for delivery are many. Some examples are genereplacement therapy, therapeutic genetic medicine for acquired diseases,polynucleotide vaccines, immunotherapy, enhanced chemotherapy and manyothers. Industrial and agricultural applications are equally varied.Some examples of industrial uses are extraction of material from cellsproduced in a fermenter, large scale transfection for production ofrecombinant protein, modification of cells for industrial use,sterilization of liquids or vaccine production. Some examples ofagricultural uses are vaccines for livestock (to include ungulates,avian species and aquatic animals) and modification of genes forimprovement of selected traits.

For standard in vitro electroporation, cuvettes are usually used. Theseare chambers that consist of parallel plate electrodes encased inplastic and have limited capacity. Volumes used in these cuvettes areunder one milliliter. The limited volume limits the total capacity fortreating cells.

Typical cell densities used are in the range of 1 million to 10 millioncells per milliliter. The cells are typically placed in a physiologicalmedium with high ionic content such as phosphate buffered saline, whichhas a conductivity of 0.017 Siemens/cm (which is equal to (17,000microSiemens/cm).

In electroporation, cell density is an important parameter. If the cellsare not dense enough, therapeutic or other material is wasted. If thecells are too dense the electric field in the proximity of each cell isnot uniform in direction or in intensity. To produce consistent resultsthat are required for clinical applications the electric fields close tothe cells must be both uniform in direction and intensity. According toFomekong et al in “Passive electrical properties of RBC suspensions:changes due to distribution of relaxation times in dependence on thecell volume fraction and medium conductivity”, in Bioelectrochemistryand Bioenergenetics, 1998, Vol 47: 81-88), the effect of cells on theelectrical properties of cell suspensions is dependent upon the packedcell volume of the cells. For packed cell volumes less than 10% thedistance between cells increases rapidly and therefore the interferingeffect of one cell to another in the electric field decreases rapidlybelow a packed cell volume of 10%. A typical cell of 15 microns indiameter would be at 10% packed cell volume at approximately 60 millioncells/ml (calculated using a mean cell volume of 0.000001767 mm3/cell).Thus cell densities under 60 million cells/ml should be used andnormally cell densities under 30 million cells/ml are used.

TABLE 1 Electrode Chamber Volume (milliliters) Number of Number of CellsOut Cells In Cell Density Million/ml Million/ml 20 million/ml 40million/ml 10 20 1 0.5 100 200 20 5 1000 2000 100 50

Clinical application generally requires 10 million to 500 million cellsin which the large molecules have been properly inserted. If a treatmentrequires 10 million cells per dose (treatment) and 5 doses are required,at least 50 million therapeutic cells must be prepared. If theefficiency of the electroporation process is assumed to be 50% and cellsare treated at a cell density of 20 million cells/ml then a 5 mlcapacity chamber would be required (50 million×2/20 million). If 100million therapeutic cells are required, a 10 ml capacity chamber wouldbe needed.

Simply increasing the size of the electrodes and the chamber to achievethe desired capacity is not practical because this causes aproportionate increase in amperage due to a decrease in resistance inthe electrodes and the suspension within the chamber. As the size of theelectrodes and the chamber increase, the resistance of the electrodesand suspension within the chamber decreases as long as the conductivityof the suspension in the chamber remains constant.

If 100 million therapeutic cells are required and the input cell densityis 20 million cells per milliliter then a 20 ml chamber is required.

In this case just scaling the size of a chamber up to 20 millilitersdoes not work. As the volume of a suspension within the chamberincreases, the resistance of the suspension within the chamberdecreases. The resistance of the suspension in the chamber is calculatedas follows:

$\begin{matrix}{R = {\frac{1}{\sigma}\frac{gap}{area}{ohms}}} & {{Formula}\mspace{14mu} 1}\end{matrix}$where s=conductivity in Siemens/cm, gap is in cm and plate area is incm². In addition:

$\begin{matrix}{{volume} = {{gap}*{area}{\mspace{11mu}\;}{cm}^{3}\mspace{14mu}{and}}} & {{Formula}\mspace{14mu} 2} \\{R = {\frac{1}{\sigma}\frac{{gap}^{2}}{volume}{ohms}}} & {{Formula}\mspace{14mu} 3}\end{matrix}$

FORMULAS 1, 2, and 3 are taken from Electroporation and Electrofusion inCell Biology, edited by Eberhard Neumann, Arthur Sowers, and CarolJordan, Plenum Press, 1989, mentioned hereinabove.

The TABLE 2 below shows the resistance of the suspension within thechamber as a function of volume for a 4-millimeter gap and media withsuspension conductivity of 0.017 Siemens/cm.

TABLE 2 Chamber Volume Media/Suspension Resistance (ml) (ohms) 0.5 19.21 9.6 5 1.92 10 0.96 50 0.19

When the chamber volume is above 1 ml, the resistance of the ionicsolution becomes impractically small; significant solution heating willoccur due to the high pulse current destroying the cells.

To address this problem a flow though technique was developed. In thisprocess the large volume of media with suspension flows through a smalltreatment chamber, and the voltage pulse waveform is applied to theparallel plates in the chamber. The problems with this process are:

1. Not all the cells are exposed to the same electric field intensityand direction.

2. There is no guarantee that the density of the material to be insertedand the cell density are constant.

3. Only uniform pulse voltages may be applied. Variable rectangularpulse waveforms such as disclosed in U.S. Pat. No. 6,010,613 cannot beused.

In a flow through process there is no guarantee that all cells will besubjected to the same electric field intensity and direction. In thisrespect, because of the properties of laminar and turbulent flow, notall of the cells will be treated for the same period of time in a flowthrough process. Lamina proximal to walls of flow through conduitstravel slower than lamina distal to the walls. Flow through processesare used in both food processing where the electric field intensity isover 20,000 volts/cm and in inserting molecules into cells fortherapeutic purposes.

A large body of prior art exists in the field of electroporation, and anumber of aspects of this body of art are of particular interest herein.For example, of particular interest herein are disclosures of theelectroporation medium, with special attention directed to mediumparameters. In this respect, TABLE 3 herein sets forth a number ofreferences relating to electroporation medium parameters such ascations, anions, osmolarity, and buffering.

TABLE 3 The following table summarizes the current state of the art:Cations Conductivity High Low Publication (μs/cm) Conc. Conc. AnionsOsmolarity Buffer Invention Low (50-150) None Ca, Mg Organic L-NHistidine 5,124,259 High K Ca, Mg Organic N 6,040,184 Very low None NoneNone L-N None 6,338,965 Very low None None None L-N None 6,368,784 HighK Ca, Mg Cl N Phos, HEPES Djuzenova Moderate to Na, K Ca Cl, N Phos.1996 high (800-14000) Sulfate Kinosita High Na Cl Phos. 1977 DimitrovLow to Na Phos., Cl Phos. 1990 Moderate Rols 1989 Low and Na Cl Phos.high Pucilar Low and Na, K (if Mg Cl, N Phos. 2001 high used) Sulfate

More particularly with respect to TABLE 3, U.S. Pat. No. 5,124,259describes an electroporation medium that provides high transfectionefficiency. The medium has potassium ions (35-105 milligramequivalents/Liter) and organic anions and is essentially devoid ofchloride ions. The medium is highly conductive as a result of thepotassium ions. The use of low conductive medium to allow the use oflarge electroporation electrodes is not discussed.

U.S. Pat. Nos. 6,040,184 and 6,338,965 describe an electroporationmedium with essentially no ions. The medium is made non-ionic throughthe use of sugars and no inorganic ions. The patent describes increasedtransfection efficiency in bacteria with the non-ionic medium. Thepatent does not mention the addition of a small amount of organic ionsto provide some conductivity and therefore some current to maintain anelectric field during electroporation.

U.S. Pat. No. 6,368,784 describes an electroporation buffer that is alsoa cryoprotectant. It also describes the use of this material forfreezing cells prior to transfection. The medium used has a highconcentration of potassium ions similar to that in intracellularcytoplasm and similar to that described in U.S. Pat. No. 5,124,259. Thepatent does not describe the use of electroporation medium with lowerconductivity to allow the use of larger capacity electrodes.

Conductivity of the medium affects the movement of material into cells.Djuezenova (Djuzenova et al, Biochemica et Biophysica Acta V 1284, 1996,p 143-152) showed that the uptake of small molecules is increased inlower conductivity medium down to 1 mS/cm, the lowest conductivity usedin the study. Others have concurred that lower conductivity increasesthe permeability of cells to small molecules during electroporation.(Kinosita, K, Tsong, T Y, Proc. Natl. Acad Sci, USA, 1977 V74:1923-1927)(Kinosita, K, Tsong, T Y Nature, 1977 V268:438-440) (Dimitrov, D S,Sowers, A E, Biochem. Biophys. Acta. 1990, V 1022:381-392).

Kinosita found that with a given electric field, media of highconductivity allowed leakage of small ions (sodium and potassium) andmedium of lower conductivity allowed passage of larger molecules(sucrose but not proteins) through red blood cell membranes. Morespecifically, Kinosita et al disclose hemolysis of human erythrocytesemploying an electroporation step. With respect to the cell used forelectroporation, there is no disclosure of electrode surface area.Therefore, and of key importance, cell chamber volume is indeterminable.A broad range of medium conductivities is stated. A broad range ofelectrode gaps is stated. Yet, there is no teaching provided forchoosing any particular set of medium conductivity and electrode gap.

Dimitrov showed that leakage of a fluorescent dye from electroporatedred blood cells was less in medium with a moderate conductivity comparedto medium with a low conductivity. Using a sensitive assay forpermeability of small molecules one group (Pucihar, G et al,Bioelectrochemistry 2001, V 54: 107-115) showed that lowering theconductivity of an electroporation buffer resulted in no change ofpermeability at given electric fields but an increase in viable cells.The assay used, electroporation using bleomycin, detects small amountsof uptake of small molecules and would not be sensitive to differencesin amount of electroporation in a given cell.

Others have found just the opposite effect, such as disclosed in “Betterpermeability of cells to small molecules was seen during electroporationusing media of higher conductivity” (Rols, M P, Tiessie, Eur. J Biochem1989 V 179:109-115). Rols and Tiessie showed that permeability to asmall molecule, Trypan Blue, was greater in high sodium medium at equalfield strength and equal number of pulses. Others (vnd den Hoff, M J,Christoffels, V M, Labruyere, W T, Moorman, A F, Lamers, W H,Electrotransfection with “intracellular” buffer, 1995, Methods Mol.Biol. V48:185-197) used high levels of potassium to mimic intracellularionic content in an effort to preserve cell viability. A more recentstudy (Baron, S et al, J. Immunol. Meth., 2000 V 242: 115-126) usedcommercially available medium with a high potassium content (VisSpan,Belzer U W cold-storage solution, DuPont Pharmaceuticals) to increaseelectroporation efficacy. The material delivered during this study wasmacromolecules such as proteins and DNA.

None of the above references discussed the use of medium with lowerconductivity to achieve the movement of macromolecules into mammaliancells. None of the references discussed the use of medium with lowerconductivity to allow the use of larger capacity electrodes.

Other components of the medium contribute both to transfection and tocell viability. One component that has been used is potassium. Potassiumin physiological levels equal to intracellular amounts tends to increaseviability in electroporated cells. This was shown by van den Hoff (vanden Hoff et al., Nucleic Acids Res., vol. 20, No. 11, 1992, p. 2902) andothers. The addition of potassium to electroporation medium increasesthe conductivity of the medium and makes the medium less desirable foruse in larger electrodes.

Calcium ions also are reported to increase viability of cells followingelectroporation. The reason for the increase in viability is reported tobe a contribution by calcium in the resealing process afterelectroporation. The increase in viability due to calcium is slightlyoffset by decreased uptake of small molecules, presumably by the samemechanism of increased pore closure due to calcium. The increase inviability due to small amounts of calcium (0.1 mM), is obtained at a lowcost in terms of increased conductivity because of the small amountused. Therefore, the addition of calcium to electroporation medium isdesirable.

Osmolarity of the medium affects cell viability and the efficiency ofmovement of large molecules through cell membranes. Most electroporationis done using media with normal osmolarity. However, the use ofhypoosmolar media can increase the efficiency of DNA transfection. (vanden Hoff et al, Nucleic Acids Res., vol. 18, No. 21, 1990, p. 6464)(Golzio et al., Biophys. J., vol. 74, 1998, pp. 3015-3022). Osmolaritycan be adjusted in electroporation media using non-ionic compounds suchas sugars, sugar alcohols, aminosugars of other non-toxic organiccompounds. These materials do not add to the conductivity. Conductivitycan be precisely controlled using inorganic anions with inorganic ororganic cations. The use of non-ionic organic material to adjustosmolarity without affecting conductivity is desirable.

Other references include:

Melkonyan et al., “Electroporation efficiency in mammalian cells isincreased by dimethyl sulfoxide (DMSO)”, Nucleic Acids Res., vol. 24,No. 21, 1996, pp. 4356-4357 and Rols et al., “Control by ATP and ADP ofvoltage-induced mammalian-cell-membrane permeabilization, gene transferand resulting expression”, Eur. J. Biochem., vol. 254, 1998, pp.382-388.

Other parameters are of interest herein with respect to electroporationmethods and apparatus disclosed in the prior art. Of particular interestare the parameters of capacity, environment for cell treatment (staticor flow), treated material, whether clinical use is provided for, andmedia or buffer used. TABLE 4 sets forth a number of U.S. patents withrespect to these parameters.

TABLE 4 Treated Media or Patent Capacity Static or flow materialClinical use buffer used 4,695,472 Large Flow Food N Food 4,695,547Small Static Cells N Any 4,838,154 Large Flow Food N Food 4,849,089Small Static Cells N Any 4,882,281 Small Static Cells N Any 5,048,404Large Flow Food N Food 5,098,843 Large Flow* Cells Possibly Non-Ionic5,128,257 Small Static Adherent cells N Ionic 5,134,070 Small StaticAdherent cells N Ionic 5,137,817 Small ** Static Cells Y Any 5,173,158Small Static (on Cells Possibly Any filter) 5,186,800 Small StaticBacteria N Low ionic 5,232,856 Small Static Adherent cells N Any5,235,905 Large Flow Food N Food 5,283,194 Small Static Cells PossiblyAny 5,545,130 Large Flow Blood Y Ionic 5,676,646 Large Flow Blood YIonic 5,720,921 Large Flow Blood Y Ionic 5,776,529 Large Flow Food NIonic 5,874,268 Small Static Adherent cells N Any 6,001,617 Small StaticAdherent cells N Any 6,074,605 Large Flow Blood Y Ionic Notes for TABLE4: *Electric field is always on, no pulses, effective pulse widthdetermined by flow rate ** Electrodes plated onto surface

More specifically with respect to the patents set forth in TABLE 2, U.S.Pat. No. 4,695,472 describes the treatment of food by electroporationusing a large volume flow-through chamber. Cannot reduce conductivity offood, has large effective capacity, no clinical use.

U.S. Pat. No. 4,695,547 describes round electrodes for electroporationwithin round tissue culture plates. No low conductive medium, no largesize, no clinical use.

U.S. Pat. No. 4,838,154 describes the treatment of food byelectroporation using a large volume flow-through chamber. Cannot reduceconductivity of food, has large effective capacity, no clinical use.

U.S. Pat. No. 4,849,089 describes round electrodes for electroporationusing fully enclosed chambers. No low conductive medium, no large size,no clinical use.

U.S. Pat. No. 4,882,281 describes round electrodes for electroporationwithin round tissue culture plates. No low conductive medium, no largesize, no clinical use.

U.S. Pat. No. 5,048,404 describes the treatment of food byelectroporation using a large volume flow-through chamber. Cannot reduceconductivity of food, has large effective capacity, no clinical use.

U.S. Pat. No. 5,098,843 describes a flow through electroporation chamberfor transfection of cells. The pulse is always on and the effectivepulse width is determined by the time in the chamber (flow rate).Non-ionic medium is described, large volume capacity, possible clinicaluse but not described.

U.S. Pat. No. 5,128,257 describes an apparatus for transfecting cellsgrown as adherent cells. Apparatus consists of multiple parallel platesplaced on a monolayer of cells. Only buffer described is PBS (highlyionic), large capacity difficult due to monolayer of cells. Clinical usenot described.

U.S. Pat. No. 5,134,070 describes a chamber for culturing cells on anoptically transparent surface that is conductive. The chamber is forelectroporation of the adherent cells. Low-ionic medium is mentioned inthe claims but no specific formula is discussed. Large capacitydifficult because of adherent cells, no clinical use mentioned.

U.S. Pat. No. 5,137,817 describes a variety of electrodes. The exampleused non-ionic medium, however it mentions that a variety of differentionic strength media can be used. In vivo and in vitro electrodes aredescribed. The in vitro electrodes are small capacity because they haveelectrodes plated onto surfaces (not easily scalable). Low ionic mediumused, small capacity, clinical uses mentioned for in vivo electrodes.

U.S. Pat. No. 5,173,158 mentions the electroporation of cells that aretrapped in pores of a non-conducting membrane. Low voltages are possiblebecause all current flows through the membrane pores. Electroporationmedium conductivity or ionic content is not mentioned. No clinical useis mentioned. Small capacity due to the need to trap cells in a pore.

U.S. Pat. No. 5,186,800 describes the transfection of prokaryotes(bacteria). Low ionic medium is used. Does not describe the use of lowionic medium with mammalian cells. States small capacity is desired. Noclinical use described.

U.S. Pat. No. 5,232,856 describes electroporation where one electrode ispartially conductive. A tilted electrode may be used on one of theelectrodes to compensate for the uneven electric fields generated usingone partially conductive electrode. Although not clear in the claims,the partially conductive electrode is for adherence of cells to itssurface. Ionic content of medium not mentioned. Adherence would limitsize. Clinical use is not mentioned.

U.S. Pat. No. 5,235,905 describes the use of electroporation to processliquid food. Large capacity flow through electrode is described. Ioniccontent of food is not adjustable. Large static capacity is notdescribed. Clinical use is not described.

U.S. Pat. No. 5,283,194 mentions the electroporation of cells that aretrapped in pores of a non-conducting membrane. Low voltages are possiblebecause all current flows through the membrane pores. Electroporationmedium conductivity or ionic content is not mentioned. No clinical useis mentioned. Small capacity due to the need to trap cells in a pore.

U.S. Pat. No. 5,514,391 describes the use of electroporation to processliquid food. Large capacity flow through electrode is described. Ioniccontent of food is not adjustable. Large static capacity is notdescribed. Clinical use is not described.

U.S. Pat. Nos. 5,545,130 and 5,676,646 describe a flow throughelectroporation device. It is designed to treat whole blood. Materialcan be added to the blood that is not ionic but blood is highly ionic.Large capacity is due to flow through. Low conductivity is not mentionedfor increasing capacity. Large static capacity is not described.Clinical use is described.

U.S. Pat. No. 5,720,921 describes a flow through electroporationchamber. A modification is made to add flexible walls to buffer pressurechanges. The main example given is to treat red blood cells byintroducing material in them that increases the release of oxygen fromthe cells. An electroporation medium is used that is conductive. Largecapacity is due to flow through. Low conductivity is not mentioned forincreasing capacity. Large static capacity is not described. Clinicaluse is described.

U.S. Pat. No. 5,776,529 describes the use of electroporation to processliquid food. Large capacity flow through electrode is described. Ioniccontent of food is not adjustable. Large static capacity is notdescribed. Clinical use is not described.

U.S. Pat. No. 5,874,268 describes an electroporation chamber designed toelectroporated adherent cells. The intent of the invention is to reducethe number of cells needed. Large capacity is not mentioned. Specificelectroporation buffers are not mentioned (just a statement about usingany electroporation buffer). Clinical use is not described.

U.S. Pat. No. 6,001,617 describes an optically transparentelectroporation chamber for treatment of adherent cells. Size is limitedby adherent cells. No low ionic medium is discussed. No clinical use isdiscussed.

U.S. Pat. No. 6,074,605 describes a flow through electroporationchamber. The main example given is to treat red blood cells byintroducing material in them that increases the release of oxygen fromthe cells. An electroporation medium is used that is conductive. Largecapacity is due to flow through. Low conductivity is not mentioned forincreasing capacity. Large static capacity is not described. Clinicaluse is described.

Another aspect of the prior art relates to the parameters ofconductivity in conjunction with electrode dimensions (height, width,and gap), presence or absence of a cuvette, volume, and dimension, suchas shown in TABLE 5.

TABLE 5 Electrode Dimensions Static, no adherent cells ElectrodeDimensions Conductivity Width Gap Cuvette Volume Publication (μs/cm)Height mm mm mm ml Dimension 5,124,259 High (~10K) 2 87.5 4 N 0.7 0.236,040,184 Very low Y 0.1-0.4 6,338,965 Very low Y 0.1-0.4 6,368,784 High(~17K) 4 0.4 Djuzenova Moderate to 6 N 1.2 0.3 1996 high (800-14000)*Kinosita Saline and  5-100 2-10 N, cross Not 1977 sucrose section =determinable 50-200 from mm{circumflex over ( )}2 publication ReimannPBS 30 30 10 0.11 1975 Dimitrov Low to 2 N 0.003 66 1990 Moderate(~100-10K) Pucilar 0.0011-1.61 S/m 2 0.05 0.8 2001 Baron High (~17K) 40.4 0.4 2000 Schwister PBS 30 30 10 N 10 0.1 1985 Mussauer 1.5-3.5 mS/cm2 0.4 0.1 2001 Mussauer 1-8 mS/cm 6 1.1 0.33 1999 Fomekong 0.064-1.447S/cm 5 0.884 0.28 1998 5,128,257 Saline 10-20 50-80 0.5-1.5 5,186,800Water 0.5-2.5 0.001-1 0.5- hundreds

Having discussed prior art above, it is clear that the foregoing body ofprior art does not teach or suggest electroporation methods andapparatus which have the following combination of desirable features:(1) can be used for clinical and therapeutic purposes wherein all cells,ex vivo or in vitro, are subject to substantially the same processconditions; (2) is scalable so that substantially large volumes of exvivo or in vitro cells can be processed in a relatively short period oftime; (3) achieves increased biological cell capacity without increasingthe size of chamber resulting in excessively large amperagerequirements; (4) limits heating within the chamber to low levels; (5)exposes substantially all ex vivo or in vitro cells to the same electricfield intensity and direction; (6) permits variable rectangular pulsewaveforms such as disclosed in U.S. Pat. No. 6,010,613 can be employed;(7) avoids problems in flow through treatment cells that are due tolaminar and turbulent flow conditions; (8) permits the use of mediumwith lower conductivity to achieve the movement of macromolecules intomammalian cells and to allow the use of larger volume chambers; and (9)is easily scalable to large capacity without using a flow throughtreatment chamber for cells to be treated.

The foregoing desired characteristics are provided by the unique largevolume ex vivo electroporation method of the present invention as willbe made apparent from the following description thereof. Otheradvantages of the present invention over the prior art also will berendered evident.

DISCLOSURE OF INVENTION

In view of the above, it is an object of the present invention is toprovide a large volume ex vivo electroporation method which can be usedfor clinical and therapeutic purposes wherein all cells, ex vivo or invitro, are subject to substantially the same process conditions.

Still another object of the present invention is to provide a largevolume ex vivo electroporation method that is scalable so thatsubstantially large volumes of ex vivo or in vitro cells can beprocessed in a relatively short period of time.

Yet another object of the present invention is to provide a large volumeex vivo electroporation method, which achieves increased biological cellcapacity without increasing the size of the chamber resulting inexcessively large amperage requirements.

Even another object of the present invention is to provide a largevolume ex vivo electroporation method that limits heating within thetreatment cell to low levels.

Still a further object of the present invention is to provide a largevolume ex vivo electroporation method which exposes substantially all exvivo or in vitro cells to the same electric field intensity anddirection.

Still another object of the present invention is to provide a largevolume ex vivo electroporation method, which permits variablerectangular pulse waveforms such as disclosed in U.S. Pat. No. 6,010,613can be employed.

Yet another object of the present invention is to provide a large volumeex vivo electroporation method that avoids problems in flow throughtreatment cells that are due to laminar and turbulent flow conditions.

Still a further object of the present invention is to provide a largevolume ex vivo electroporation method that permits the use of mediumwith lower conductivity to achieve the movement of macromolecules intomammalian cells and to allow the use of larger capacity chambers.

Yet another object of the present invention is to provide a large volumeex vivo electroporation method, which is easily scalable to largecapacity without using a flow through treatment chamber for cells to betreated.

These together with still other objects of the invention, along with thevarious features of novelty, which characterize the invention, arepointed out with particularity in the claims annexed to and forming apart of this disclosure. For a better understanding of the invention,its operating advantages and the specific objects attained by its uses,reference should be had to the accompanying drawings and descriptivematter in which there are illustrated preferred embodiments of theinvention.

To achieve the foregoing and other advantages, the present invention,briefly described, provides a static chamber with large volume to insureall cells are subject to the same electric field intensity and directionand the density of the cells and material are uniform. With thisinvention any waveform may be used. This invention is a voltage waveformgenerator connected to an electrode with parallel plates with has lowconductivity media, a cell density of 20 million cells per 10milliliters or less. The invention uses media with conductivity between50 μS/cm and 500 μS/cm (which is equal to 50 microSiemens/cm to 500microSiemens/cm). The invention may be used in clinical applications andhas a closed sterile chamber into which the cells and large moleculesare inserted and removed.

In accordance with one aspect of the invention, a method is provided oftreating vesicles with exogenous material for insertion of the exogenousmaterial into the vesicles includes the steps of:

a. retaining a suspension of the vesicles and the exogenous material ina treatment volume in a chamber which includes electrodes, wherein thechamber has a geometric factor (cm⁻¹) defined by the quotient of theelectrode gap squared (cm²) divided by the chamber volume (cm³), whereinthe geometric factor is less than or equal to 0.1 cm⁻¹), wherein thesuspension of the vesicles and the exogenous material is in a mediumwhich is adjusted such that the medium has conductivity in a rangespanning 50 microSiemens/cm to 500 microSiemens/cm, wherein thesuspension is enclosed in the chamber during treatment, and

b. treating the suspension enclosed in the chamber with one or morepulsed electric fields,

wherein in accordance with a. and b. above, the treatment volume of thesuspension is scalable, and wherein the time of treatment of thevesicles in the chamber is substantially uniform.

Preferably, the chamber is a closed chamber. Preferably, the chamber hasat least a 2 milliliter capacity. The chamber and the contents thereofcan be sterile. Preferably, the chamber includes entry and exit portsfor entry and removal of the suspension. Preferably, the electrodes areparallel plate electrodes.

The electric fields are substantially uniform throughout the treatmentvolume. The electric fields can include a rectangular voltage pulsewaveform to produce a uniform pulse electric field between parallelplate electrodes greater than 100 volts/cm and less than 5,000 volts/cm,substantially uniform throughout the treatment volume.

The vesicles can be living cells, i.e., eukaryotes and prokaryotes, andthe medium can be a physiological medium and has a conductivity between50 and 500 μS/cm (which equals 50 microSiemens/cm to 500microSiemens/cm). The number of living cells that are treated in thechamber at one time can be more than 10 million in number. Furthermore,the number of living cells that are treated in the chamber at one timecan be more than 20 million in number.

The vesicles can be autologous cells that are to be returned to a donorafter treatment with the exogenous material. The vesicles can besyngeneic cells that are to be given to a recipient other than thedonor. The vesicles can be zenogeneic cells. The vesicles can beartificial liposomes.

The pulsed electric fields can be from electrical pulses, which are in asequence of at least three non-sinusoidal electrical pulses, havingfield strengths equal to or greater than 100 V/cm, to the material. Thesequence of at least three non-sinusoidal electrical pulses has one,two, or three of the following characteristics (1) at least two of theat least three pulses differ from each other in pulse amplitude, (2) atleast two of the at least three pulses differ from each other in pulsewidth, and (3) a first pulse interval for a first set of two of the atleast three pulses is different from a second pulse interval for asecond set of two of the at least three pulses.

With the method of the invention, the temperature rise during vesicletreatment is miniscule.

The method of the invention, with respect to the chamber volume, isscalable in a range spanning 2 to 10 milliliters. The method of theinvention can be carried out in sequential batches.

The exogenous material can be a therapeutic material. The exogenousmaterial can be a therapeutic product formed from the treatment of thevesicles with exogenous material. The exogenous material can be selectedfrom the following group: a polynucleotide; DNA; RNA; a polypeptide; aprotein; and an organic compound.

The exogenous material can include numerous base pairs, for example, atleast eight base pairs.

With the invention, the chamber has a chamber volume, the suspension hasa suspension volume, and the suspension volume is greater than thechamber volume. In this respect, an initial portion of the suspensionvolume is moved into the chamber, retained and treated in the chamber,and moved out from the chamber. Then, an additional portion of thesuspension volume is moved into the chamber, retained and treated in thechamber, and moved out from the chamber.

Still further portions of the suspension volume are sequentially movedinto the chamber, retained and treated in the chamber, and moved outfrom the chamber. These steps can be repeated until the suspensionvolume is depleted.

In accordance with another aspect of the invention, an electroporationapparatus is provided which includes a chamber which has a chambervolume of at least 2 milliliters. A pair of electroporation electrodesare contained within the chamber. An electroporation medium, carryingvesicles in suspension, is contained in the chamber between theelectroporation electrodes. The medium has a conductivity between 50microSiemens/cm and 500 microSiemens/cm. A source of pulsed voltages iselectrically connected to the electroporation electrodes, and means foradding material to the chamber for electroporation treatment therein.Also, means are provided for removing treated material from the chamber.

Preferably, sealing means are connected to the chamber for providing asealed chamber. The sealing means can include a quantity of elastomermaterial.

Preferably, the sealed chamber is sterile inside the chamber.Preferably, the chamber includes vent means for venting air when fluidis moved into the chamber. The vent means can include a filter member ina wall of the chamber. Alternatively, the vent means can include a ventcell in fluid communication with the chamber.

The chamber includes a chamber inlet and a chamber outlet. A firstreservoir can be provided in fluid communication with the chamber inlet,for containing the vesicle-bearing electroporation medium prior tointroduction into the chamber. A second reservoir can be provided influid communication with the chamber inlet, for containing a chamberflushing material for flushing treated vesicle-bearing medium out fromthe chamber. A third reservoir can be provided in fluid communicationwith the chamber outlet, for receiving treated, vesicle-bearing mediumthat is flushed out from the chamber.

The first reservoir, the second reservoir, and the third reservoir canbe comprised of flexible bags. An inlet valve can be connected betweenthe chamber inlet and the first reservoir and the second reservoir, andan outlet valve can be connected between the chamber outlet and thethird reservoir.

BRIEF DESCRIPTION OF DRAWINGS

The invention will be better understood and the above objects as well asobjects other than those set forth above will become more apparent aftera study of the following detailed description thereof. Such descriptionmakes reference to the annexed drawing wherein:

FIG. 1A is a schematic illustration of apparatus employed with carryingout the method of the invention.

FIG. 1B is a schematic illustration of the electroporation chamber withits parts of the electroporation apparatus.

FIG. 2 is a graph illustrating the operating range of the method of theinvention, inside the triangle, and how the operating range of theinvention is outside operating ranges of prior art electroporationmethods, indicated by small blocks outside the triangle.

FIG. 3 is a graph illustrating the relationship between charging time(in microseconds) of biological cells and media conductivity (inmicroSiemens/cm) for cells having three different diameters, namely 1micrometer, 10 micrometers, and 100 micrometers.

FIG. 4 is a graph showing Time Constant versus Conductivity as itrelates to the method of the invention.

MODES FOR CARRYING OUT THE INVENTION

As previously described a significant problem is the conductivity of themedia used in electroporation. In the process of the invention, a lowconductivity medium is employed to keep the total resistance of thesuspension greater than one ohm, wherein heating in the chamber islimited to low levels. Not just any medium conductivity can be used. Asthe ionic content of the medium is reduced, the number of free ions thatare available to build charge (voltage) across the cell member isdecreased. The effect is to increase the amount of time it takes tocharge the membrane. This process is described by the equation inElectroporation and Electrofusion in Cell Biology, edited by EberhardNeumann, Arthur Sowers, and Carol Jordan, Plenum Press, 1989, on page71. Assuming a typical cell diameter of 10 microns, the charging time is20 microseconds at 80 μS/cm. Below 80 μS/cm the charging time become toolong and the pathways in cell membrane stop forming. The TABLE 6 belowillustrates the resistance of the media as a function of electrodechamber volume and conductivity.

TABLE 6 Chamber Suspension Resistance - ohms Volume ml 17,000 μS/cm 200μS/cm 80 μS/cm 0.5 19.2 160 0   4000 1 9.6 800 2000 5 1.92 160 400 100.96  80 200 50 0.19  16 40

Ex vivo electroporation has been demonstrated in numerous publishedresearch projects. At this point commercial applications, such asclinical transfection to produce a vaccine for the patient, requireslarge electrodes or chambers to process millions of cells at one time.The static parallel plate chamber provides the most uniform amplitudeand most uniform electric field direction of any configurationavailable. This uniformity is required to insure uniform treatment ofthe target cells. It is also important not to use very high-density cellconcentration such as 30 million cells/ml to insure local uniformelectric fields about the cells. This invention applies to chambers in arange spanning 2 to 10 milliliters.

Using larger chambers results in high current flow when voltage isapplied. The equations for chamber resistance vs. conductivity of thecell and media mixture and the chamber dimensions are as follows:

Volume  of  material = l × A${{Resistance}\mspace{14mu}{of}\mspace{14mu}{Material}} = {{\rho\frac{l}{A}} = {{\frac{1}{\sigma}\frac{l}{A}} = {{\frac{1}{\sigma}\frac{l^{2}}{\upsilon}} = {\frac{G\; F}{\sigma}{ohms}}}}}$

-   ρ=resistivity in ohm-cm-   σ=1/ρ in Siemens/cm-   υ=volume of material being treated-   l=gap between electrodes (cm)-   A=area of electrode (cm²)

There is a Geometric Factor (GF), which is a constant for any chamberdimension. As the volume of the chamber gets larger the resistance ofthe material in the chamber gets smaller thus increasing current flow.

The present invention uses an electrode with large capacity incombination with an electroporation buffer of defined low conductivity.This process exposes all cells to the same treatment conditions,provides control over the amperage required and can process largenumbers of cells. Since the cell suspension statically remains in thechamber during application of pulsed electric fields, complex waveformscan be used.

Another aspect of the invention further increases capacity byalternately filling and emptying the gap between the electrodes. In thismanner, all desired properties are met during a specific treatment andthe electrodes can be re-used for subsequent treatments in a sequentialbatch process.

This present invention specifies a range of medium and suspensionconductivities, which can be used versus the chamber dimensions, thelarger the volume the smaller the conductivity. This invention specifiesan operating area for use with the larger volume chambers. This isillustrated in FIG. 2 . Operating points of prior art published resultsare also presented in FIG. 2 as squares. For chambers with a GeometricFactor less than 0.1 there are two limiting factors, which are related.The first is the absolute value of the chamber resistance. In thisinvention the chamber resistance is one ohm or greater. Operating belowone ohm is viewed as impractical. The other constraint is theconductivity of the medium and suspension in the chamber. As theconductivity decreases the charging time of the cell membrane increasesbecause there are fewer ions external to the cell membrane.

The relationship between the Transmembrane Voltage (TMV) andconductivity and cell diameter is as follows, taken from Newman et alstated below:Transmembrane Voltage=TMVTMV=−1.5Er|cos δ|f(λ)

-   -   where: E=electric filed in volts/cm        -   r=cell radius in cm        -   δ=angle from electric field line in degrees

f(λ) = composite  conductivity${f(\lambda)} = \frac{\lambda_{0}{\lambda_{1}\left( {2\frac{d}{r}(\;)} \right.}}{{\left( {{2\;\lambda_{o}} + \lambda_{i}} \right)\lambda_{m}} + {\left( {2\frac{d}{r}} \right)\left( {\lambda_{o} - \lambda_{m}} \right)\left( {\lambda_{i} - \lambda_{m}} \right)}}$

-   -   where: λ_(o)=conductivity of media external to cell        milliSiemens/cm        -   λ_(i)=conductivity of cytoplasm        -   λ_(m)=conductivity of cell membrane        -   d=thickness of cell membrane

-   Reference: Electroporation and Electrofusion in Cell Biology    -   Edited by Eberhard Neumann, Arthur Sowers, and Carol Jordon    -   Plenum Press, 1989

Below 1 microSiemens/cm there are so few ions that the time to changethe cell membrane is unrealistically large.

The preferred operating region of the present invention is then:

-   Cell diameter>1 micrometer-   Chamber volume 2 to 10 milliliters-   Conductivity of Material to be treated 50 microSiemens/cm to 500    microSiemens/cm-   Total resistance of material to be treated in chamber>1 ohm-   Geometric Factor of Chamber<0.1 cm⁻¹

The invention uses a static chamber with large volume to insure that allcells in suspension are subject to the same electric field intensity anddirection and the density of the cells and treating material areuniform. With this invention any waveform may be used. This inventionincludes a voltage waveform generator connected to electrodes in theform of parallel plates, and, which has a low conductivity medium and asuspension in the static chamber, having a cell density of 20 millioncells or less.

A component of the invention is the use of low conductivity mediumwithin a defined range to limit amperage and heat while simultaneouslyproviding enough ions to effectively electroporate cells. Typically themedium used will have a conductivity in a range spanning 50microSiemens/cm to 500 microSiemens/cm.

The invention may be used in clinical applications and with a closedsterile chamber into which the cells and large molecules are insertedand removed.

One aspect of the invention further increases capacity by alternatelyfilling and emptying the chamber. In this manner, all desired propertiesare met during a specific treatment and the chamber can be re-used forsubsequent treatments in a sequential batch process.

The conductivity of the medium used in electroporation is an importantaspect of this invention. In this process, a low conductivity medium isemployed to keep the total resistance of the medium small and virtuallyeliminate heating. There is a limit to the lower conductivity mediumthat can be used. As the ionic content of the medium is reduced thenumber of free ions that are available to build charge (voltage) acrossthe cell membrane is decreased. The effect is to increase the amount oftime it takes to charge the membrane. This process is described by theequation in Neumann, p 71. Assuming a typical cell diameter of 10microns, the charging time is 20 microseconds at a conductivity of 80microSiemens/cm. For a typical cell diameter of 10 microns, below 80microSiemens/cm, the charging time becomes too long and the pathways incell membranes stop forming.

Using an electrode with a 4 mm gap, TABLE 6 illustrates the resistanceof the medium as a function of electrode chamber volume andconductivity.

In one aspect of the invention, a chamber with two electrodes is used asshown in FIG. 1 . An example of electrode dimensions that can be used isa gap of 0.4 cm, electrode height of 2 cm and electrode length of 10 cm.The chamber can be used with a commercial electroporator such as theCyto Pulse Sciences, Inc. PA-4000 electroporator.

An example of a medium that can be used with the chamber is one with thefollowing formula:

-   Sorbitol 280 millimoles-   Calcium Acetate, 0.1 millimoles-   Magnesium Acetate, 0.5 millimoles

FIG. 3 is a graph illustrating the relationship between charging time(in microseconds) of biological cells and media conductivity (inmicroSiemens/cm) for cells having three different diameters, namely 1micrometer, 10 micrometers, and 100 micrometers. From FIG. 3 it is clearthat for media conductivity below 1 microSiemen/cm, the charging timewould be so large that electroporation would not work.

As to the manner of usage and operation of the instant invention, thesame is apparent from the above disclosure, and accordingly, no furtherdiscussion relative to the manner of usage and operation need beprovided.

It is apparent from the above that the present invention accomplishesall of the objects set forth by providing a large volume ex vivoelectroporation method which may advantageously be used for clinical andtherapeutic purposes wherein all cells, ex vivo or in vitro, are subjectto substantially the same process conditions. With the invention, alarge volume ex vivo electroporation method is provided which isscalable from 2 to 10 milliliters so that substantially large volumes ofex vivo or in vitro cells can be processed in a relatively short periodof time. With the invention, a large volume ex vivo electroporationmethod is provided which achieves increased biological cell capacitywithout increasing the size of a chamber resulting in excessively largeamperage requirements. With the invention, a large volume ex vivoelectroporation method is provided which limits heating within thechamber to low levels. With the invention, a large volume ex vivoelectroporation method is provided which exposes substantially all exvivo or in vitro cells to the same electric field intensity anddirection. With the invention, a large volume ex vivo electroporationmethod provides that the density of the material to be inserted into thetreatment chamber can be held constant. With the invention, a largevolume ex vivo electroporation method is provided which permits variablerectangular pulse waveforms such as disclosed in U.S. Pat. No. 6,010,613can be employed. With the invention, a large volume ex vivoelectroporation method is provided which avoids problems in flow throughtreatment cells that are due to laminar and turbulent flow conditions.With the invention, a large volume ex vivo electroporation method isprovided which permits the use of medium with lower conductivity toachieve the movement of macromolecules into mammalian cells and to allowthe use of larger capacity chambers. With the invention, a large volumeex vivo electroporation method is provided which is easily scalable tolarge capacity without using a flow through treatment chamber for cellsto be treated.

The electroporation chamber with its parts 1 as shown by FIG. 1Bincludes the electroporation chamber 13 with a geometric factor definedby the quotient of the electrode gap squared (cm²) divided by thechamber volume (cm³), wherein the geometric factor is less than or equalto 0.1 cm⁻¹. The chamber 13 has an inlet 2 and an outlet 3 configuredfor sequential passage of suspensions and fluids during electroporation.The chamber 13 includes a first reservoir 4 in fluid communication withthe inlet 2 for containing the suspensions and fluids prior tointroducing into the chamber 13 for treatment, a second reservoir 5 influid communication with the inlet 2 for containing flushing materialfor flushing the suspensions and fluids after treatment out from thechamber 13, and a third reservoir 6 in fluid communication with theoutlet 3 for receiving the suspensions and fluids flushed aftertreatment out from the chamber 13. The inlet valve 10 is connectedbetween the inlet 2 and the first reservoir 4 and the inlet valve 11 isconnected between the second reservoir 5 and inlet 2, and the outletvalve 12 is connected between the outlet 9 and the third reservoir 6.The chamber 13 includes the vent 7 for removing air when the chamber isfilled with a fluid. The chamber includes sealing means 9 for providinga sealed chamber. The chamber 13 includes parallel plate electrodes 8.Not shown by FIG. 1B, the chamber 13 includes a source of pulsedvoltages electrically connected to said parallel plate electrodes 8configured to provide pulse waveforms and an uniform electric fieldbetween the parallel plate electrodes 8, wherein the uniform electricfield is greater than 100 volts/cm and less than 5,000 volts/cm andsubstantially uniform throughout the chamber 13.

With respect to the above description, it should be realized that theoptimum dimensional relationships for the parts of the invention, toinclude variations in size, form function and manner of operation,assembly and use, are deemed readily apparent and obvious to thoseskilled in the art, and therefore, all relationships equivalent to thoseillustrated in the drawings and described in the specification areintended to be encompassed only by the scope of appended claims.

While the present invention has been shown in the drawings and fullydescribed above with particularity and detail in connection with what ispresently deemed to be the most practical and preferred embodiments ofthe invention, it will be apparent to those of ordinary skill in the artthat many modifications thereof may be made without departing from theprinciples and concepts set forth herein. Hence, the proper scope of thepresent invention should be determined only by the broadestinterpretation of the appended claims so as to encompass all suchmodifications and equivalents.

What is claimed is:
 1. An apparatus for electroporation comprising: asterile electroporation chamber with a geometric factor defined by thequotient of the electrode gap squared (cm²) divided by the chambervolume (cm³), wherein the geometric factor is less than or equal to 0.1cm⁻¹; wherein the chamber is configured for sequential electroporationand the chamber has an inlet and an outlet configured for sequentialpassage of suspensions and fluids during electroporation; the chambercomprising: a first reservoir in fluid communication with the inlet forcontaining the suspensions and fluids prior to introducing into thechamber for treatment, a second reservoir in fluid communication withthe inlet for containing flushing material for flushing the suspensionsand fluids after treatment out from the chamber, and a third reservoirin fluid communication with the outlet for receiving the suspensions andfluids after treatment flushed out from the chamber; a vent for removingair when the chamber is filled with a fluid; parallel plate electrodes;and a source of pulsed voltages electrically connected to said parallelplate electrodes configured to provide pulse waveforms and an uniformelectric field between the parallel plate electrodes, wherein theuniform electric field is greater than 100 volts/cm and less than 5,000volts/cm and substantially uniform throughout the chamber.
 2. Theapparatus of claim 1, wherein the vent is a filter member in the wall ofthe chamber.
 3. The apparatus of claim 1, wherein the vent is avent-cell in fluid communication with the chamber.
 4. The apparatus ofclaim 1, wherein the chamber further comprises an elastomeric seal. 5.The apparatus of claim 1, wherein the first reservoir contains a lowconductivity medium for suspending vesicles for electroporation, whereinthe conductivity of the medium is between 50 microSiemens/cm and 500microSiemens/cm.
 6. The apparatus of claim 1, wherein the volume of thechamber is between 2 ml and 10 ml.
 7. The apparatus of claim 1, whereinthe first reservoir, the second reservoir, and the third reservoircomprise flexible bags.
 8. The apparatus of claim 1, wherein an inletvalve is connected between the chamber inlet and the first reservoir andthe second reservoir, and an outlet valve is connected between thechamber outlet and the third reservoir.